Miyamoto C M, Graham A D, Boylan M, Evans J F, Hasel K W, Meighen E A, Graham A F
J Bacteriol. 1985 Mar;161(3):995-1001. doi: 10.1128/jb.161.3.995-1001.1985.
DNA coding for the alpha and beta subunits of Vibrio harveyi luciferase, the luxA and luxB genes, and the adjoining chromosomal regions on both sides of these genes (total of 18 kilobase pairs) was cloned into Escherichia coli. Using labeled DNA coding for the alpha subunit as a hybridization probe, we identified a set of polycistronic mRNAs (2.6, 4, 7, and 8 kilobases) by Northern blotting; the most prominent of these was the one 4 kilobases long. This set of mRNAs was induced during the development of bioluminescence in V. harveyi. Furthermore, the same set of mRNAs was synthesized in E. coli by a recombinant plasmid that contained a 12-kilobase pair length of V. harveyi DNA and expressed the genes for the luciferase subunits. A cloned DNA segment corresponding to the major 4-kilobase mRNA coded for the alpha and beta subunits of luciferase, as well as a 32,000-dalton protein upstream from these genes that could be specifically modified by acyl-coenzyme A and is a component of the bioluminescence system. V. harveyi mRNA that was hybridized to and released from cloned DNA encompassing the luxA and luxB genes was translated in vitro. Luciferase alpha and beta subunits and the 32,000-dalton polypeptide were detected among the products, along with 42,000- and 55,000-dalton polypeptides, which are encoded downstream from the lux genes and are thought to be involved in luminescence.
哈维氏弧菌荧光素酶的α和β亚基的编码DNA(luxA和luxB基因)以及这些基因两侧相邻的染色体区域(共18千碱基对)被克隆到大肠杆菌中。用编码α亚基的标记DNA作为杂交探针,我们通过Northern印迹法鉴定出一组多顺反子mRNA(2.6、4、7和8千碱基);其中最突出的是4千碱基长的那一种。这组mRNA在哈维氏弧菌生物发光发育过程中被诱导产生。此外,同一组mRNA在大肠杆菌中由一个含有12千碱基对哈维氏弧菌DNA的重组质粒合成,并表达荧光素酶亚基的基因。一个与主要的4千碱基mRNA相对应的克隆DNA片段编码荧光素酶的α和β亚基,以及这些基因上游的一种32000道尔顿的蛋白质,该蛋白质可被酰基辅酶A特异性修饰,是生物发光系统的一个组成部分。与包含luxA和luxB基因的克隆DNA杂交并从其中释放的哈维氏弧菌mRNA在体外进行翻译。在产物中检测到了荧光素酶α和β亚基以及32000道尔顿的多肽,还有42000和55000道尔顿的多肽,它们由lux基因下游编码,被认为与发光有关。